Novel reagents and method for conjugating biological molecules

ABSTRACT

A compound of the general formula X—[NR—CO—Ar—CO—(CH═CH) n —(CH 2 ) 2 -L] m  (I) in which X represents a polymer; R represents a hydrogen atom or an alkyl, aryl or alkyl-aryl group; Ar represents an unsubstituted or substituted heteroaryl or aryl group; n represents 0 or an integer of from 1 to 4; L represents a leaving group; and m represents an integer of from 1 to 8. The compounds find use in the conjugation of biological molecules.

This invention relates to novel reagents and a novel method forconjugating biological molecules, especially proteins and peptides.

Many therapeutically active molecules, for example proteins, do notpossess the properties required to achieve efficacy in clinical medicaluse. For example, many native proteins do not make good medicinesbecause upon administration to patients there are several inherentdrawbacks that include: (1) proteins are digested by many endo- andexo-peptidases present in blood or tissue; (2) many proteins areimmunogenic to some extent; and (3) proteins can be rapidly excreted bykidney ultrafiltration and by endocytosis. Some molecules which mightfind utility as active therapeutic agents in medicines are systemicallytoxic or lack optimal bioavailability and pharmacokinetics. Whenproteins clear from the blood circulation quickly they typically have tobe administered to the patient frequently. Frequent administrationfurther increases the risk of toxicity, especially immunologicallyderived toxicities.

Water soluble, synthetic polymers, particularly polyalkylene glycols,are widely used to conjugate therapeutically active molecules such asproteins, peptides and low molecular weight drugs. These therapeuticconjugates have been shown to alter pharmacokinetics favourably byprolonging circulation time and decreasing clearance rates, decreasingsystemic toxicity, and in several cases, displaying increased clinicalefficacy. The process of covalently conjugating polyethylene glycol,PEG, to proteins is commonly known as “PEGylation”.

It is important for optimised efficacy and to ensure dose to doseconsistency that the number of conjugated polymer molecules per proteinis the same for each molecule, and that each polymer molecule isspecifically covalently conjugated to the same amino acid residue ineach protein molecule. Non-specific conjugation at sites along a proteinmolecule results in a distribution of conjugation products and,frequently, unconjugated protein, to give a complex mixture that isdifficult and expensive to purify.

For thiol specific conjugation, PEGylation reagents having a PEG chainterminated on one end with a maleimide group are commonly used. Suchreagents are described in many publications, for example WO 2004/060965.Maleimide-terminated reagents are commercially available. However, manyPEG-maleimides are hydrolytically unstable during storage andconjugation to a drug candidate. Specifically, a substantial degree ofhydrolysis of the maleimide ring occurs, both prior to and afterconjugation.

We have now found a class of PEGylation reagents which can be used toconjugate molecules including proteins and peptides to polymers via asingle nucleophilic residue, for example a thiol group, and which haveadvantages, over such commercial reagents.

Accordingly, the present invention provides a process for theconjugation of a molecule containing a thiol or amino group to apolymer, which comprises reacting said molecule with a compound of thegeneral formula:

X-[Q-W—(CH═CH)_(n)—(CH₂)₂-L]_(m)  (I)

in which X represents a polymer; Q represents a linking group; Wrepresents an electron-withdrawing group; n represents 0 or an integerof from 1 to 4; L represents a leaving group; and m represent an integerof from 1 to 8.

The direct product resulting from the process of the invention may begenerally represented by the general formula:

X-[Q-W—(CH═CH)_(n)—(CH₂)₂—Z]_(m)  (IIa)

in which X, Q, W n and m have the meanings given above, and Z representssaid molecule conjugated via a thiol or amino group. If desired, thisresulting compound of formula IIa can be converted into any otherdesired product. Specifically, a resulting compound of the generalformula IIa may be converted into a compound of the general formula II

X-[Q-W′—(CH═CH)_(n)—(CH₂)₂—Z]_(m)  (II)

in which W′ represents an electron-withdrawing moiety or a moietypreparable by reduction of an electron-withdrawing moiety.

The compounds of the general formula (I) are novel, and the inventiontherefore also provides these compounds per se. Particularly preferrednovel compounds of the formula (I) are those of the formula (Ia) asdefined below.

The compounds of the general formula (II) are also novel, and theinvention therefore also provides these compounds per se. Particularlypreferred novel compounds of the formula (II) are those of the formula(IIb) as defined below.

m represents an integer from 1 to 8, for example 1 to 6, preferably 1 to4, for example 1. Where m is 1, a single molecule is conjugated to thepolymer. When m is greater than one, the conjugation of more than onemolecule to a polymer may be accomplished. From 2 to 8 groups-Q-W′—(CH═CH)_(n)—(CH₂)₂—Z or -Q-W—(CH═CH)_(n)—(CH₂)₂-L are attached tothe polymer, and the variables Q, W, W′, n, L and Z may be the same ordifferent for each such group. Multi-functional polymer compounds areavailable, for example, multiple groups may be attached using asstarting material the multi-arm compounds available from NOF under theTrade Mark “Sunbright”: e.g. the 4-arm products have the formulaC[CH₂O(CH₂CH₂O)n-Y]₄ where Y may be one of a number of different endgroups. Multimeric conjugates can result in synergistic and additivebenefits. For example, if m is 1, the resulting conjugate must have anend group on the end of the PEG chain remote from the conjugatedmolecule. This is commonly an alkoxy or similar group, and it has beensuggested that such groups may lead to undesired immunogenic effectswhen used for pharmaceutical applications. If m is greater than 1, aconjugated molecule can be attached to both ends of the PEG chain,dispensing with the need for an end group such as alkoxy.

A polymer X may for example be a polyalkylene glycol, apolyvinylpyrrolidone, a polyacrylate, for example polyacryloylmorpholine, a polymethacrylate, a polyoxazoline, a polyvinylalcohol, apolyacrylamide or polymethacrylamide, for examplepolycarboxymethacrylamide, or a HPMA copolymer. Additionally X may be apolymer that is susceptible to enzymatic or hydrolytic degradation. Suchpolymers, for example, include polyesters, polyacetals, poly(orthoesters), polycarbonates, poly(imino carbonates), and polyamides, such aspoly(amino acids). A polymer X may be a homopolymer, random copolymer ora structurally defined copolymer such as a block copolymer. For exampleX may be a block copolymer derived from two or more alkylene oxides, orfrom poly(alkylene oxide) and either a polyester, polyacetal, poly(orthoester), or a poly(amino acid). Polyfunctional polymers that may be usedinclude copolymers of divinylether-maleic anhydride and styrene-maleicanhydride.

Naturally occurring polymers may also be used, for examplepolysaccharides such as chitin, dextran, dextrin, chitosan, starch,cellulose, glycogen, poly(sialylic acid) and derivatives thereof. Aprotein may be used as the polymer. This allows conjugation of oneprotein, for example an antibody or antibody fragment, to a secondprotein, for example an enzyme or other active protein. Also, if apeptide containing a catalytic sequence is used, for example an O-glycanacceptor site for glycosyltransferase, it allows the incorporation of asubstrate or a target for subsequent enzymatic reaction. Poly(aminoacid)s such as polyglutamic acid or polyglycine may also be used, as mayhybrid polymers derived from natural monomers such as saccharides oramino acids and synthetic monomers such as ethylene oxide or methacrylicacid.

If the polymer is a polyalkylene glycol, this is preferably onecontaining C₂ and/or C₃ units, and is especially a polyethylene glycol.A polymer, particularly a polyalkylene glycol, may contain a singlelinear chain, or it may have branched morphology composed of many chainseither small or large. The so-called Pluronics are an important class ofPEG block copolymers. These are derived from ethylene oxide andpropylene oxide blocks. Substituted polyalkylene glycols, for examplemethoxypolyethylene glycol, may be used. In a preferred embodiment ofthe invention, a single-chain polyethylene glycol is initiated by asuitable group, for example an alkoxy, e.g. methoxy, aryloxy, carboxy orhydroxyl group, and is connected at the other end of the chain to thelinker group Q.

The polymer X may optionally be derivatised or functionalised in anydesired way. Reactive groups may be linked at the polymer terminus orend group, or along the polymer chain through pendent linkers; in suchcase, the polymer is for example a polyacrylamide, polymethacrylamide,polyacrylate, polymethacrylate, or a maleic anhydride copolymer. Suchfunctionalised polymers provide a further opportunity for preparingmultimeric conjugates (i.e. conjugates in which the polymer, isconjugated to more than one molecule). If desired, the polymer may becoupled to a solid support using conventional methods.

The optimum molecular weight of the polymer will of course depend uponthe intended application. Preferably, the number average molecularweight is in the range of from 250 g/mole to around 75,000 g/mole. Whenthe compound of the general formula II is intended for medical use andis intended to leave the circulation and penetrate tissue, for examplefor use in the treatment of inflammation caused by malignancy, infectionor autoimmune disease, or by trauma, it may be advantageous to use alower molecular weight polymer in the range 2000-30,000 g/mole. Forapplications where the compound of the general formula II is intended toremain in circulation it may be advantageous to use a higher molecularweight polymer, for example in the range of 20,000-75,000 g/mole.

The polymer to be used should be selected so the conjugate is soluble inthe solvent medium for its intended use. For biological applications,particularly for diagnostic applications and therapeutic applicationsfor clinical therapeutic administration to a mammal, the conjugate willbe soluble in aqueous media. However, many biological molecules, forexample proteins such as enzymes, have utility in industry, for exampleto catalyze chemical reactions. For conjugates intended for use in suchapplications, it may be necessary that the conjugate be soluble ineither or both aqueous and organic media. The polymer should of coursenot unduly impair the intended function of the molecule to beconjugated.

Preferably the polymer is a synthetic polymer, and preferably it is awater-soluble polymer. The use of a water-soluble polyethylene glycol isparticularly preferred for many applications.

A linking group Q may for example be a direct bond, an alkylene group(preferably a C₁₋₁₀ alkylene group), or an optionally-substituted arylor heteroaryl group, any of which may be terminated or interrupted byone or more oxygen atoms, sulphur atoms, —NR groups (in which Rrepresents a hydrogen atom or an alkyl (preferably C₁₋₆alkyl), aryl(preferably phenyl), or alkyl-aryl (preferably C₁₋₆alkyl-phenyl) group),keto groups, —O—CO— groups, —CO—O— groups, —O—CO—O, —O—CO—NR—,—NR—CO—O—, —CO—NR— and/or —NR.CO— groups. Such aryl and heteroarylgroups Q form one preferred embodiment of the invention. Suitable arylgroups include phenyl and naphthyl groups, while suitable heteroarylgroups include pyridine, pyrrole, furan, pyran, imidazole, pyrazole,oxazole, pyridazine, primidine and purine. Especially suitable linkinggroups Q are heteroaryl or, especially, aryl groups, especially phenylgroups, terminated adjacent the polymer X by an —NR.CO— group. Thelinkage to the polymer may be by way of a hydrolytically labile bond, orby a non-labile bond.

Substituents which may be present on an optionally substituted aryl orheteroaryl group include for example one or more of the same ordifferent substituents selected from alkyl (preferably C₁₋₄alkyl,especially methyl, optionally substituted by OH or CO₂H), —CN, —NO₂,—CO₂R, —COH, —CH₂OH, —COR, —OR, —OCOR, —OCO₂R, —SR, —SOR, —SO₂R, —NHCOR,—NRCOR, NHCO₂R, —NR.CO₂R, —NO, —NHOH, —NR.OH, —C═N—NHCOR, —C═N—NR.COR,—N⁺R₃, —N⁺H₃, —N⁺HR₂, —N⁺H₂R, halogen, for example fluorine or chlorine,—C≡CR, —C═CR₂ and —C═CHR, in which each R independently represents ahydrogen atom or an alkyl (preferably C₁₋₆alkyl), aryl (preferablyphenyl), or alkyl-aryl (preferably C₁₋₆alkyl-phenyl) group. The presenceof electron withdrawing substituents is especially preferred. Preferredsubstituents include for example CN, NO₂, —OR, —OCOR, —SR, —NHCOR,—NR.COR, —NHOH and —NR.COR.

W may for example represent a keto group CO, an ester group —O—CO— or asulphone group —SO₂—, and W′ may represent such a group or a groupobtained by reduction of such a group, e.g. a CH.OH group, an ethergroup CH.OR, an ester group CH.O.C(O)R, an amine group CH.NH₂, CH.NHR orCH.NR₂, or an amide CH.NHC(O)R or CH.N(C(O)R)₂.

Preferably n is 0.

A leaving group L may for example represent —SR, —SO₂R, —OSO₂R, —N⁺R₃,—N⁺HR₂, —N⁺H₂R, halogen, or —OØ, in which R has the meaning given above,and Ø represents a substituted aryl, especially phenyl, group,containing at least one electron withdrawing substituent, for example—CN, —NO₂, —CO₂R, —COH, —CH₂OH, —COR, —OR, —OCOR, —OCO₂R, —SR, —SOR,—SO₂R, —NHCOR, —NRCOR, —NHCO₂R, —NR′CO₂R, —NO, —NHOH, —NR′OH,—C═N—NHCOR, —C═N—NR′COR, —N⁺R₃, —N⁺HR₂, —N⁺H₂R, halogen, especiallychlorine or, especially, fluorine, —C≡CR, —C═CR₂ and —C═CHR, in whicheach R independently has one of the meanings given above.

If m represents an integer of from 2 to 8, different L groups may bepresent if desired. This provides an opportunity, by selecting L groupsof different reactivity, to conjugate different molecules to the polymerX in successive reactions.

Preferably the process according to the invention uses a reagent of thegeneral formula:

X—[NH—CO—Ar—W—(CH═CH)_(n)—(CH₂)₂—SO₂R′]_(m)  (Ia)

in which Ar represents an unsubstituted or substituted aryl group,especially a phenyl group, in which the optional substituents areselected from those mentioned above for an aryl group contained inlinking group Q; R′represents a hydrogen atom or an optionallysubstituted alkyl (preferably C₁₋₆alkyl), aryl (preferably phenyl), oralkyl-aryl (preferably C₁₋₆alkyl-phenyl) group; and W and m have themeanings given above; to produce a novel conjugate of the generalformula:

X—[NH—CO—Ar—W′—(CH═CH)_(n)—(CH₂)₂—Z]_(m)  (IIb)

In these preferred compounds Ia and IIb, preferably n is 0, andpreferably m represents an integer of from 1 to 4, especially 1.Preferably each of W represents a CO group, and W′ represents a CO groupor a CH.OH group. Preferably R′ represents an optionally substitutedalkyl group, for example an optionally hydroxy substituted C₁₋₄alkylgroup such as —CH₂CH₂OH, or, especially, a C₁₋₄alkyl-aryl group,especially p-tolyl. Preferably Ar is an unsubstituted phenyl group.Preferably X is a polyalkylene glycol, especially polyethylene glycol.

The compounds of formula I, and especially those of formula Ia, arebelieved to be surprisingly stable, and also have high reactivitytowards molecules containing a thiol or amino group. It is believed thatthe conjugation process according to the invention proceeds via theformation of an intermediate compound of the general formula

X-[Q-W—(CH═CH)_(n)—CH═CH₂]_(m)  V

in which X, Q, W, n and m have the meanings given above.

The molecule to be conjugated by the process of the present inventionmay be any desired molecule. It may for example be a naturally-occurringmolecule or a molecule derived from a naturally occurring molecule, orit may be any molecule having biological activity, for example any drug,provided that it contains a thiol (—SH) or amino (—NHR) group. Forexample, it may be a protein or a peptide: throughout thisspecification, the term “protein”, will be used for convenience, andexcept where the context requires otherwise, references to “protein”should be understood to be references to “protein or peptide”.

The thiol or amino group through which the molecule is linked is anucleophile capable of reacting with the reagent I, with elimination ofthe leaving group L. Such groups may be present in a native biologicalmolecule, or may be introduced into a biological molecule prior toconjugation.

Two thiol groups may be generated by reduction of a natural orengineered disulfide (cysteine) bridge, which may be intrachain orinterchain. A biological molecule can contain one or a multiplicity ofdisulfide bridges, and reduction to give free sulfhydral moieties can beconducted to reduce one or a multiplicity of disulfide bridges.Depending on the extent of disulfide reduction and the stoichiometry ofthe polymeric conjugation reagent that is used, it is possible toconjugate one or a multiplicity of polymer molecules to the biologicalmolecule. Immobilised reducing agents may be used if it is desired toreduce less than the total number of disulfides, as can partialreduction using different reaction conditions or the addition ofdenaturants.

Alternatively a thiol group can be a single cysteine residue or otherthiol group not originally derived from a disulfide bridge. A singlecysteine may be introduced by synthetic means to provide a suitablepoint of attachment. Such procedure is particularly useful for theconjugation of peptides.

Amine groups may for example be lysine or histidine residues. These maybe present in a native biological molecule, or introduced synthetically.For example, a histidine residue might be introduced by way of ahis-tag, a short chain of contiguous histidine residues, for examplecontaining up to 12 histidine residues but typically containing 5 or 6residues, attached by synthetic methods to a protein. His-tags bindstrongly to nickel and cobalt, enabling them to be bound to a nickel- orcobalt-containing column used in the separation process known asimmobilized-metal affinity chromatography. His-tags are widely used,being attached to a wide range of proteins and peptides to enable themor products derived therefrom to be separated from mixtures at a futuredate.

Where the molecule is a protein, it may for example be a peptide,polypeptide, antibody, antibody fragment, enzyme, cytokine, chemokine,receptor, blood factor, peptide hormone, toxin, transcription protein,or multimeric protein.

The following gives some specific proteins which may have utility in thepresent invention, depending upon the desired application. Enzymesinclude carbohydrate-specific enzymes, proteolytic enzymes and the like.Enzymes of interest, for both industrial (organic based reactions) andbiological applications in general and therapeutic applications inparticular include the oxidoreductases, transferases, hydrolases,lyases, isomerases and ligases disclosed by U.S. Pat. No. 4,179,337.Specific enzymes of interest include asparaginase, arginase, adenosinedeaminase, superoxide dismutase, catalase, chymotrypsin, lipase,uricase, bilirubin oxidase, glucose oxidase, glucuronidase,galactosidase, glucocerbrosidase, glucuronidase, and glutaminase.

The proteins used in compounds of the general formula I include forexample blood proteins such as albumin, transferring, Factor VII, FactorVIII or Factor IX, von Willebrand factor, insulin, ACTH, glucagen,somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentaryhormones, somatomedins, erythropoietin, luteinizing hormone,hypothalamic releasing factors, antidiuretic hormones, prolactin,interleukins, interferons, colony stimulating factors, hemoglobin,cytokines, antibodies, antibody fragments, chorionicgonadotropin,follicle-stimulating hormone, thyroid stimulating hormone and tissueplasminogen activator.

Certain of the above proteins such as the interleukins, interferons andcolony stimulating factors also exist in non-glycosylated form, usuallythe result of preparation by recombinant protein techniques. Thenon-glycosylated versions may be used in the present invention.

Other proteins of interest are allergen proteins disclosed by Dreborg etal Crit. Rev, Therap. Drug Carrier Syst. (1990) 6 315-365 as havingreduced allergenicity when conjugated with a polymer such aspoly(alkylene oxide) and consequently are suitable for use as toleranceinducers. Among the allergens disclosed are Ragweed antigen E, honeybeevenom, mite allergen and the like.

Glycopolypeptides such as immunoglobulins, ovalbumin, lipase,glucocerebrosidase, lectins, tissue plasminogen activator andglycosilated inerleukins, interferons and colony stimulating factors areof interest, as are immunoglobulins such as IgG, IgE, IgM, IgA, IgD andfragments thereof.

Of particular interest are receptor and ligand binding proteins andantibodies and antibody fragments which are used in clinical medicinefor diagnostic and therapeutic purposes. The antibody may be used aloneor may be covalently conjugated (“loaded”) with another atom or moleculesuch as a radioisotope or a cytotoxic/antiinfective drug. Epitopes maybe used for vaccination to produce an immunogenic polymer-proteinconjugate.

Biological molecules may be derivatised or functionalised if desired. Inparticular, prior to conjugation, the molecule, for example a nativeprotein, may have been reacted with various blocking groups to protectsensitive groups thereon; or it may have been previously conjugated withone or more polymers or other molecules, either using the process ofthis invention or using an alternative process.

The molecule may be a synthetic molecule of relatively low molecularweight. It may, for example, be any drug for which conjugation providesadvantages. For many such molecules, it will be necessary to insert asuitable linker carrying thiol or amino group to enable it to beconjugated to the reagent according to the invention. Typical drugsinclude for example captopril, amphotericin B, camptothecin, taxol,irinotecan and its derivatives such as SN38, docetaxol, and ribavirin.

Other molecules of interest which may be conjugated using the process ofthe invention include those listed in WO 2004/060965.

The process according to the invention may be carried out in a solventor solvent mixture in which all reactants are soluble. The molecule tobe conjugated, for example a protein, may be allowed to react directlywith the compound of the general formula I in an aqueous reactionmedium. This reaction medium may also be buffered, depending on the pHrequirements of the nucleophile (thiol or amine). The optimum pH for thereaction will in many cases be at least 6.0, typically between about 6.8and about 8.5, for example about 7.0 to 8.0, preferably about 7.5-8.0,but in other cases pH's of as low as 4.0 may be used, particularly whenconjugation to a polyhistidine tag is required, leading to a usable pHrange of from 4.0 to 8.5. If it is preferred to generate the compound ofthe formula V above in situ in the presence of the molecule to beconjugated, a relatively high pH is suitably used throughout.Alternatively, if it is preferred to generate the compound of theformula V above in a separate step and subsequently add the molecule tobe conjugate, the first step is suitably carried out at a relativelyhigh pH (e.g. 7-5-8.0) while the subsequent step is suitably carried outat a lower pH (e.g. 6.0 to 6.5). It is an advantage of the reagents ofthe present invention that they may be used successfully over arelatively wide range of pH conditions.

Reaction temperatures between 3-37° C. are generally suitable: proteinsmay decompose, aggregate or denature impairing function and/or reactionefficiency if the conjugation reaction is conducted at a temperaturewhere these processes may occur. Reactions conducted in organic media(for example THF, ethyl acetate, acetone) are typically conducted attemperatures up to ambient.

The molecule can be effectively conjugated with the desired reagentusing a stoichiometric equivalent or a slight excess of reagent, unlikemany other reagents. However, since the reagents do not undergocompetitive reactions with aqueous media used to solvate for exampleproteins, it is possible to conduct the conjugation reaction with anexcess stoichiometry of reagent. The excess reagent and the product canbe easily separated by ion exchange chromatography during routinepurification, or by separation using nickel if a his-tag is present.

Where the group through which the molecule is conjugated is a thiolgroup, the process according to the invention may be carried out bypartially reducing a disulfide bond derived from two cysteine aminoacids in the biological molecule in situ following which the reducedproduct reacts with the compound of formula (I). Disulfides can bereduced, for example, with dithiothreitiol, mercaptoethanol, ortris-carboxyethylphosphine using conventional methods.

The immediate product of the process according to the invention is acompound of the general formula II in which W′ is anelectron-withdrawing group. Such compounds have utility in themselves:because the process of the invention is reversible under suitableconditions, compounds of formula (II) in which W′ is anelectron-withdrawing moiety have utility in applications where releaseof the molecule from the conjugate is required, for example in directclinical applications. An electron-withdrawing moiety W′ may, however,be reduced to give a moiety which prevents release of the molecule, andsuch compounds will also have utility in many clinical, industrial anddiagnostic applications.

Thus, for example, a moiety W′ containing a keto group may be reduced toa moiety W′ containing a CH(OH) group; an ether group CH.OR may beobtained by the reaction of a hydroxy group with an etherifying agent;an ester group CH.O.C(O)R may be obtained by the reaction of a hydroxygroup with an acylating agent; an amine group CH.NH₂, CH.NHR or CH.NR₂may be prepared from a ketone or aldehyde by reductive amination); or anamide CH.NHC(O)R or CH.N(C(O)R)₂ may be formed by acylation of an amine.The use of a borohydride, for example sodium borohydride, sodiumcyanoborohydride, potassium borohydride or sodium triacetoxyborohydride,as reducing agent is particularly preferred. Other reducing agents whichmay be used include for example tin(II) chloride, alkoxides such asaluminium alkoxide, and lithium aluminium hydride.

A compound of the general formula (I) may be prepared by reacting acompound of the general formula

A-Q-W—(CH═CH)_(n)—(CH₂)₂-L  (III)

in which Q, W, n and L have the meanings given above, with a compound ofthe general formula

X—B_(m)  (IV)

in which X represents a polymer; A and B being groups selected such thatthe compounds of (III) and (IV) will react together to give the desiredcompound of the formula (I).

The compounds of the general formula II have a number of applications.They may for example be used for direct clinical application to apatient, and accordingly, the present invention further provides apharmaceutical composition comprising a novel compound of the generalformula II together with a pharmaceutically acceptable carrier. Theinvention further provides a novel compound of the general formula IIfor use in therapy, and a method of treating a patient which comprisesadministering a pharmaceutically-effective amount of a novel compound ofthe formula II or a pharmaceutical composition according to theinvention to the patient. Any desired pharmaceutical effect, for exampletrauma treatment, enzyme replacement, protein replacement, woundmanagement, toxin removal, anti-inflammatory, anti-infective,immunomodulatory, vaccination or anti-cancer, may be obtained bysuitable choice of biological molecule. Compounds of the general formulaII may include an imaging agent, for example a radio-nucleotide, toenable tracking of the compound in vivo.

The compounds of the general formula II may also be used in non-clinicalapplications. For example, many physiologically active compounds such asenzymes are able to catalyse reactions in organic solvents, andcompounds of the general formula II may be used in such applications.Further, compounds of the general formula II may be used as diagnostictools.

The following Examples illustrate the invention. In the accompanyingdrawings:

FIG. 1 shows the reaction scheme of step 1 of Example 1.

FIG. 2 shows the reaction scheme of step 2 of Example 1.

FIG. 3 shows the reaction scheme of step 3 of Example 1.

FIG. 4 shows the reaction scheme of step 4 of Example 1.

FIG. 5 shows the result of an SDS-PAGE analysis of the product ofExample 2.

FIG. 6 shows the result of an SDS-PAGE analysis of the product ofExample 3.

FIG. 7 shows the result of an SDS-PAGE analysis of the product ofExample 5.

FIG. 8 shows reverse-phase chromatography analysis from the reaction inExample 6.

FIG. 9 shows the result of an SDS-PAGE analysis of the product ofExample 7.

FIG. 10 shows the cation exchange chromatography analysis from thereaction in Example 8.

FIG. 11 shows the ELISA result of Example 8.

FIG. 12 shows the result of an SDS-PAGE analysis, of the product ofExample 9.

FIG. 13 shows chromatograms of the time-course PEGylation of Example 11.

FIG. 14 shows the interconversion of compounds 9, 10 and 11 as referredto in Examples 10 and 11.

FIG. 15 shows the conversion results of Example 11.

FIG. 16 shows the reverse-phase chromatography result of Example 12.

FIG. 17 shows the stability results of Example 13.

FIG. 18 shows the result of an SDS-PAGE analysis of the intermediateproduct of Example 17.

FIG. 19 shows the result of an SDS-PAGE analysis of the final product ofExample 17.

FIG. 20 shows the absorbance results of Example 17.

FIG. 21 shows the reaction scheme of Example 19.

FIG. 22 shows the reaction scheme of Example 20.

EXAMPLE 1 PEG Reagent Synthesis: Synthesis of 10 kDa PEG Reagent 9 Step1, Synthesis of p-carboxy-3-piperidinopropriophenone hydrochloride 2

The reaction scheme for this step is shown in FIG. 1.

A 250 ml round bottom flask was charged with p-acetylbenzoic acid (15.0g, 1), 11.11 g piperidine hydrochloride and 8.23 g paraformaldehyde.Absolute ethanol (90 ml) and concentrated hydrochloric acid (1 ml) werethen added, and the resulting suspension heated under reflux for 10 hwhile stirring under argon. After stopping the reflux, acetone (150 ml)was added and the reaction mixture allowed to cool to room temperature.The resulting white precipitate was isolated on a glass filter (G3) andwashed twice with chilled acetone. The solid was then dried under vacuumto give a white crystal powder (2, 9.72 g). ¹H NMR (400 MHz, DMSO-d₆) δ1.79, 2.96, 3.45 (br m, CH₂ of piperidine moiety), 3.36 (t, 2H, COCH₂),3.74 (t, 2H, NCH₂), 8.09 (m, 4H, ArH).

Step 2: Synthesis of 4-(3-(p-tolylthio)propanoyl)benzoic acid 5

The reaction scheme for this step is shown in FIG. 2.

The p-carboxy-3-piperidinopropriophenone hydrochloride 2 (1.0 g) and4-methylbenzenethiol (417 mg, 3) were suspended in a mixture of absoluteethanol (7.5 ml) and methanol (5 ml). Piperidine (50 μl) was then addedand the suspension heated to reflux with stirring for 6 h in an argonatmosphere. The white precipitate afforded after cooling to roomtemperature was filtered off with a glass filter (G3), washed carefullywith cold acetone and dried in vacuum to give 5 (614 mg). ¹H NMR (400MHz, DMSO-d₆) δ 2.27 (s, 3H, phenyl-CH₃), 3.24, 3.39 (t, 2×2H, CH₂),7.14, 7.26 (d, 2×2H, ArH of tolyl moiety), 8.03 (m, 4H, ArH ofcarboxylic acid moiety).

Step 3, Synthesis of 4-(3-tosylpropanoyl)benzoic acid 6

The reaction scheme for this step is shown in FIG. 3.

4-(3-(p-tolylthio)propanoyl)benzoic acid 5 (160 mg) was suspended in amixture of water (10 ml) and methanol (10 ml). After cooling in an icebath, oxone (720 mg, Aldrich) was added and the reaction mixture allowedto warm to room temperature while stirring overnight (15 h). Theresulting suspension was diluted with further water (40 ml) so that itbecame nearly homogeneous and the mixture was then extracted three timeswith chloroform (in total 100 ml). The pooled chloroform extracts werewashed with brine and then dried with MgSO₄. Evaporation of volatilesunder vacuum at 30° C. afforded a white solid 6 (149 mg). ¹H NMR (400MHz, DMSO-d₆) δ 2.41 (s, 3H, phenyl-CH₃), 3.42 (t, 2H, CO—CH₂), 3.64 (t,2H, SO₂—CH₂), 7.46, 7.82 (d, 2×2H, ArH of tolyl moiety), 8.03 (m, 4H,ArH of carboxylic acid moiety).

Step 4, Synthesis of PEGylated 4-(3-tosylpropanoyl)benzoic acid, PEGreagent 9

The reaction scheme for this step is shown in FIG. 4.

The 4-(3-tosylpropanoyl)benzoic acid 6 (133 mg) andO-(2-aminoethyl)-O′-methyl-PEG 8 (MW 10 kDa, 502 mg, BioVectra) weredissolved in dry toluene (5 ml). The solvent was removed under vacuumwithout heating and the dry solid residue was then redissolved in drydichloromethane (15 ml) under argon. To the resulting solution, cooledin an ice bath, was slowly added diisopropylcarbodiimide (DIPC, 60 mg)under argon. The reaction mixture was then kept stirring at roomtemperature overnight (15 h). Volatiles were then removed under vacuum(30° C., water bath) to afford a solid residue that was redissolved withgentle heating (35° C.) in acetone (20 ml). The solution was filteredover non-absorbent cotton wool to remove insoluble material. Thesolution was then cooled in a dry ice bath to give a white precipitatethat was separated by centrifugation (4600 rpm, 30 min). The liquidphase was decanted off and this precipitation procedure was repeatedthree times. Afterwards the resulting off-white solid was dried undervacuum to afford the PEG reagent 9 (437 mg). ¹H NMR (400 MHz, CDCl₃) δ2.46 (s, 3H, phenyl-CH₃), 3.38 (s, 3H, PEG-OCH₃), 3.44-3.82 (br m, PEG),7.38, 7.83 (d, 2×2H, ArH of tolyl moiety), 7.95 (m, 4H, ArH ofcarboxylic acid moiety).

Analogous PEG reagents of different PEG molecular weights were preparedby the same general procedure. Thus, 20 kDa PEG was prepared by reactionof the sulfone 6 (20.8 mg), O-(2-aminoethyl)-O′-methyl-PEG (20 kDa, 250mg, Fluka) and DIPC (8.7 mg, 7) in dry dichloromethane (15 ml) affordingafter the acetone precipitation purification procedure an off-whitesolid (245 mg). ¹H NMR (400 MHz, CDCl₃) δ 2.46 (s, 3H, phenyl-CH₃), 3.38(s, 3H, PEG-OCH₃), 3.44-3.82 (br m, PEG), 7.38, 7.83 (d, 2×2H, ArH oftolyl moiety), 7.95 (m, 4H, ArH of carboxylic acid moiety).

EXAMPLE 2 PEGylation of a Fab Antibody Fragment Possessing a SingleHinge Disulfide (Two Thiols) with PEG Reagents 9 of Molecular Weight 5,10 and 20 kDa

To 100 μl of a Fab solution (Abcam cat. no. AB6520, 1 mg/ml) was added 5μl of a DTT stock solution (100 mM in deionised water) and the resultingsolution allowed to stand at room temperature for 30 min. The solutionwas diluted to 200 μl with 95 μl of pH 7.8, 50 mM phosphate buffer with0.15M NaCl and 10 mM EDTA, and then loaded on an illustra NAP-5 column(GE Healthcare cat. No. 17-0854-01), pre-equilibrated with pH 7.8, 50 mMphosphate buffer with 0.15M NaCl and 10 mM EDTA. The NAP-5 column waseluted with 5×300 μl of fresh pH 7.8 phosphate buffer. The UV absorbanceat 280 nm was measured for all the fractions whereupon reduced Fab wasidentified to be mainly in fraction 3 and the protein concentrationestimated to be 0.23 mg/ml.

Three PEG reagents with molecular weights 5 kDa, 10 kDa and 20 kDa weredissolved in pH 7.8 phosphate buffer to give 0.5 mg/ml, 1 mg/ml and 2mg/ml solution concentrations respectively.

For each PEGylation reaction, 5.0 μl of the reduced Fab solution (0.23mg/ml) and 0.42 μl of PEG solution (1 molar equivalent to reduced hingedisulfide thiols) was used. The Fab reaction solutions were also dilutedwith 4.6 μl of pH 7.8 phosphate buffer to give a final volume of 10 μl.The reaction′ solutions were incubated at 4° C. for 12 h.

SDS-PAGE analysis was then carried out on the reaction solutions usingNuPAGE® Novex 4-12% Bis-Tris gels Invitrogen cat. No. NP0321BOX) andNuPAGE MES SDS running buffer (Invitrogen cat. No. NP002). The gels werestained with InstantBlue (Expedeon cat. No. ISB1L). FIG. 5 below showsthe result. PEG on SDS-PAGE analysis runs approximately double its truesize against protein markers, so a 5 kDa PEG runs like a 10 kDa protein.Fab is a protein of approximately 50 kDa and on a SDS-PAGE gel runseither as a single band at about 50 kDa when non-reduced or as a band ortwo bands at around 25 kDa for the reduced form. These are the heavy andlight chains which are no longer held together by the hinge disulfideupon reduction and incubation with SDS. Therefore, although the Fab canbe di-PEGylated whereby a single PEG is attached to each of the twocysteines of the reduced hinge disulfide in solution, SDS-PAGE analysisshows mono-PEGylation of the 25 kDa heavy and the light chains. In thelane labelled 1 are the protein markers (Novex sharp protein standards,Invitrogen cat. No. LC5800). Lane 2 shows Fab itself. The lane labelled4 shows Fab reduced with DTT (around 25 kDa). Lane 5 shows the 5 kDaPEGylation result with the primary product a band at 35 kDacorresponding to PEGylated Fab. Only a small amount of reduced Fabremains at 25 kDa showing the Fab was mostly PEGylated. Lane 6 shows the10 kDa PEGylation result with the primary product a band above the 40kDa marker corresponding to 10 kDa PEGylated Fab. Lane 7 shows the 20kDa result with the primary product a band above the 60 kDa markercorresponding to 20 kDa PEGylated Fab. Lane 3 shows the result of anattempted PEGylation without a previous reduction step: the fact thatthere is no PEGylated product indicates that the reaction site forPEGylation is a reduced disulfide bond.

EXAMPLE 3 PEGylation of Asparaginase with 5 kDa PEG Reagent 9

To 1 ml of a 1 mg/ml solution of asparaginase (Sigma cat. no A3809) in20 mM sodium phosphate buffer containing 150 mM NaCl and 5 mM EDTA at pH7.8, was added DTT (15.4 mg) and after vortexing for several seconds theresulting solution was left at room temperature for 40 min. The 1 ml ofsolution was then added to a PD-10 column (GE Healthcare cat. No.17-0851-01) pre-equilibrated with pH 7.8 20 mM sodium phosphate buffer,containing 150 mM NaCl and 5 mM EDTA, collecting the eluent as the loadfraction. The column was then eluted with 5×1 ml of fresh phosphatebuffer. Fractions 3 and 4 were combined to give 2 ml.

A 10 mg/ml solution of 5 kDa PEG reagent 9 was prepared in deionisedwater and 2.0 μl (1.5 equivalents of PEG to free thiols) was added to100 μl of the reduced asparaginase. The solution was vortexed forseveral seconds and then placed at 4° C. overnight, whereafter a samplewas taken for SDS-PAGE analysis. The result is shown in FIG. 6. Lane 1shows the protein markers used to estimate MW, lane 2 shows asparaginasebefore PEGylation and lane 3 shows the reaction solution of reducedasparaginase with 5 kDa PEG reagent. A strong band corresponding to thePEGylation of both cysteines is seen as the primary product just abovethe 60 kDa protein marker. A second lower MW band is present just abovethe 50 kDa protein marker which corresponds to PEGylation of only one ofthe two cysteines. There is only a very faint band corresponding toreduced asparaginase between the 30 and 40 kDa protein markers showingthat nearly all of the protein had PEGylated with PEG reagent 9.

EXAMPLE 4 PEGylation of G-CSF (Granulocyte-Colony Stimulating Factor)Possessing a Free Single Cysteine with PEG Reagent 9 with MolecularWeights of 5, 10 and 20 kDa and Comparison with a 5 kDa PEG Possessing aMaleimide Functional Group

A GCSF stock solution (0.66 mg/ml in 50 mM sodium phosphate buffer, pH6.2, with 150 mM NaCl and 10 mM EDTA) was divided into 5 fractions of100 μL each (Native G-CSF and four fractions for the PEGylationreactions). The fractions were incubated overnight at 4° C. with 1 molarequivalent of PEG reagent to G-CSF. For 5 kDa PEG reagent 9 and for 5kDa PEG maleimide (Fluka cat. no. 63187), this involved adding 3.5 μl ofa 5 mg/ml PEG solution in deionised water. For 10 kDa PEG reagent 9 thisinvolved adding 7 μl of a 5 mg/ml PEG solution in deionised water. For20 kDa PEG reagent 9 this involved adding 14.0 μl of a 5 mg/ml solutionin deionised water. The PEGylation reactions were analysed by SDS-PAGE(NuPAGE® Novex 4-12% Bis-Tris gels, MES buffer, all from Invitrogen, andInstant-Blue stain (Expedeon cat. No. ISB1L). G-CSF without PEG reagentadded showed as a band between the 15 and 20 kDa protein markers. Forthe 5 kDa PEGylation with 9 a band at around 30 kDa corresponding to 5kDa monoPEGylated GCSF was visible. For the 10 kDa PEGylation with 9 aband below 40 kDa was visible corresponding to 10 kDa monoPEGylatedG-CSF. For the 20 kDa PEG result a band between 50 and 60 kDacorresponding to 20 kDa monoPEGylated G-CSF was visible. For the 5 kDaPEG maleimide reaction no band was seen other than unreacted G-CSF,showing that no reaction had occurred with this reagent.

EXAMPLE 5 PEGylation on Histidine in IFN α-2b with an 8 HistidineSequence on its C Terminal with 10 kDa and 20 kDa PEG Reagent 9

To a 20 μl solution of IFN α-2b (1.13 mg/ml in 10 mM sodium phosphatebuffer containing 2 mM EDTA and 150 mM NaCl, pH 7.5) was added 1 molarequivalent of 10 kDa PEG reagent 9 (1.8 μl of a 6 mg/ml solution indeionised water) and the resulting solution incubated overnight at roomtemperature. A repeat was also performed with 1 molar equivalent of 20kDa PEG reagent 9 (3.3 μl of a 6.6 mg/ml solution in deionised water.Both samples were then analysed by SDS-PAGE (NuPAGE® Novex 4-12%Bis-Tris gels, MES running buffer, all from Invitrogen, and InstantBluestain (Expedeon cat. No. ISB1L)). The result is shown in FIG. 7. In thelane labelled 1 are the protein markers. Lane 2 is the starting IFNonly. Lane 3 shows the result of the 10 kDa PEG reagent 9 reaction.There are 5 distinct bands between the 30 and 160 kDa protein markerscorresponding to IFN with 1 to 5 PEG chains conjugated. Lane 4 shows theresult of the 20 kDa PEG reagent 9 reaction. There are three distinctbands between the 60 to 110 kDa protein markers corresponding to IFNwith 1 to 3 PEG chains conjugated. The lane labelled 5 is the 20 kDa PEGreagent which does not stain, so no band is visible. The lane labelled 6is the 10 kDa PEG reagent which does not stain, so no band is visible.

EXAMPLE 6 PEGylation of a Peptide (Leptin Fragment, with a Single FreeCysteine in the Structure) with 5 kDa PEG Reagent 9

Leptin fragment 116-130 amide mouse (Sigma cat. no. L6788) 1 mg wasdissolved in 1 ml of 50 mM sodium phosphate buffer containing 150 mMNaCl and 10 mM EDTA at pH 7.8. A 5 mg/ml solution of 5 kDa PEG reagent 9was prepared in the same buffer at pH 7.8. To a 50 μl of the leptinfragment solution was added 50 μl buffer and 96.1 μl of the PEG solution(3 molar equivalents of PEG to free thiol on cysteine). The solution wasvortexed for several seconds and then placed at 4° C. overnight,whereafter samples were taken for RP-HPLC analysis. The RP-HPLCconsisted of an analytical column Source 5RPC 4.6/150 (Amershambioscience cat. no. 17511601) attached to a JASCO HPLC system. Buffer Awas water+0.05% trifluoroacetic acid (Fisher scientific HPLC grade) andbuffer B was acetonitrile (Fisher scientific HPLC grade). The method was100% to 0% of buffer A over 30 minutes with a flow rate of 1 ml/min. TheHPLC profile was monitored under 214 nm and 280 nm. Results for theleptin fragment, PEG reagent and the reaction solution are shown in FIG.8.

The leptin fragment had a retention time of 11.4 min. In the reactionmixture this peak disappeared and was replaced with a peak at 16.5 min,showing that the fragment had successfully been derivatised.

EXAMPLE 7 PEGylation and Biological Activity of a Polyhistidine TaggedDomain Antibody Fragment with PEG Reagent 9 of Molecular Weight 20 kDa

To 2.7 ml of an anti-TNF alpha domain antibody fragment solution(protein sequence taken from patent WO 2005/035572 as the sequencelisted as TAR1-5-19 in FIG. 12 and expressed with a six-histidine tag onthe C-terminus of the protein, 1.5 mg/ml in 50 mM sodium phosphate, 150mM sodium chloride and 350 mM imidazole, pH 7.5) was added 0.3 ml of asolution of PEG reagent in deionised water (40 mg/ml, 1.9 molarequivalents to protein). The solution was transferred into D-Tube™dialyzer (Novagen, cat. No. 71508-3) and dialysed against 1 L pH 6.2buffer (50 mM sodium phosphate, 150 mM sodium chloride, 20 mM EDTA) at4° C. over 16 h. The reaction solution was purified on a Resource Scolumn (GE Healthcare, cat. No. 17-1178-01) using a linear gradient over30 min from 20 mM sodium acetate, pH 4.5, to 20 mM sodium acetate with700 mM sodium chloride, pH 4.5.

Fractions were collected and analysed using SDS-PAGE and the result isshown in FIG. 9. NuPAGE® Novex 4-12% Bis-Tris gels Invitrogen cat. No.NP0321BOX) and NuPAGE MES SOS running buffer (Invitrogen cat. No. NP002)were used and the gels were stained with InstantBlue (Expedeon cat. No.ISB1L). PEG on SDS-PAGE analysis runs approximately double its true sizeagainst protein markers, so a 20 kDa PEG runs like a 40 kDa protein. Thedomain antibody fragment is a protein of approximately 12.7 kDa. In thelane labelled 1 are the protein markers (Novex sharp protein standards,Invitrogen cat. No. LC5800). Lane 2 shows the protein only. The lanelabelled 3 is the reaction solution. A band at 53 kDa corresponds tomonoPEGylated protein. DiPEG-protein is represented by a band about 80kDa and there is trace amount of multiPEGylated protein at the top ofthe lane. Lane 4 shows the purified monoPEGylated domain antibodyfragment as a single band with no contamination from unPEGylatedprotein.

EXAMPLE 8 PEGylation and ELISA Binding of an Anti-TNF Alpha Affibody (1Free Thiol Cysteine) with 20 kDa PEG Reagent 9

To 1 ml of a 1 mg/ml solution of anti-TNF alpha Affibody (Affibody ABcat. no 10.0841.01) in 50 mM sodium phosphate buffer containing 150 mMNaCl and 20 mM EDTA at pH 7.8, was added DTT (3.0 mg) to reduce anydisulfide bonds and after vortexing for several seconds the resultingsolution was left at room temperature for 30 min. The 1 ml of solutionwas then loaded to a PD-10 column (GE Healthcare cat. No. 17-0851-01)pre-equilibrated with pH 6.2 phosphate buffer (50 mM sodium phosphate,150 mM NaCl and 20 mM EDTA). The column was then eluted with 5×1 ml offresh pH 6.2 phosphate buffer. A280 nm measurements indicated fractions3 and 4 contained the reduced protein and were combined to give 2 ml. Tothe protein solution was added 10 μl of a saturated aqueous hydroquinonesolution and mixed well. A 20 mg/ml solution of 20 kDa PEG reagent 9 wasprepared in deionised water and 73 μl (1 molar equivalent of PEG to freecysteine thiol) was added to the DTT treated affibody. The solution wasvortexed for several seconds and then placed at ambient temperature for3 h, whereafter a sample was taken for SDS-PAGE analysis.

After the 3 h, a significant amount of PEGylated protein had alreadyformed so the PEGylation reaction was purified without allowing thereaction to go to completion. Purification was achieved using a ResourceS cation exchange chromatography column (GE Healthcare, cat. No.17-1178-01) with a linear salt gradient (0-700 mM NaCl) over 30 min withpH 4.5, 20 mM sodium acetate mobile phase. The result is shown in FIG.10. The lane labelled 1 in FIG. 10 shows the protein markers used toestimate MW. The lane labelled 2 shows the affibody solution beforetreatment with DTT. The affibody in the presence of DTT is shown in thelane labelled 3. Lane 4 shows the affibody after removal of the DTT byPD-10 column and two bands are visible corresponding to monomericaffibody (free thiol cysteine available for PEGylation) and dimericaffibody (cysteines not available for PEGylation). The lane labelled 5shows the PEGylation reaction solution. A band just above the 50 kDaprotein marker can be seen for the mono PEGylated affibody. The cationexchange chromatography purified monoPEGylated affibody is shown in lane6 as a single band with no contamination from unPEGylated protein.

The Binding Activity of the Purified Mono PEGylated Product to TNF-α wasAnalysed by an ELISA Method:

TNF-α (10 ug/ml in 15 mM Na₂CO₃, 34.9 mM NaHCO₃, pH 9.6) was added to a96-well microtitre plate (Maxisorp, Nunc) at 100 μl/well and incubatedat 4° C. overnight. TNF-α was then removed and PBS/1% BSA was added at100 μl/well and incubated for 1 h at RT. PBS/1% BSA was then removed andthe PEGylated anti-TNF-α affibody (0.026, 0.13, 0.65, 3.25 ug/ml inPBS/1% BSA) added and incubated for 1 h at RT. No PEGylated affibody wasadded to the control wells. The plate was then washed three times with300 μl/well of PBS/0.1% Tween 20 (PBS/T). Anti-PEG rabbit antibody(Epitomics, cat no. 2061-1; 1:1000 in 1% BSA/PBS) was then added at 100μl/well and incubated for 1 h at RT. Following three washes with PBS/T,horseradish peroxidase-conjugated anti-rabbit antibody (Abcam, cat no.ab6721; 1:1000 in PBS/1% BSA) was added at 100 μl/well and incubated for1 h at RT. Wells were then washed three times with PBS/T and3,3′,5,5′tetramethylbenzidine substrate (Sigma-Aldrich, cat no. T0440)was added at 100 μl/well. After 15 min incubation in the dark, stopreagent (Sigma-Aldrich, cat no. S5689) was added at 100 μl/well and theabsorbance read at 650 nm.

The ELISA result shown in FIG. 11 shows specific binding to TNF-α by thePEGylated affibody, confirming that the protein retains activitypost-PEGylation. In the absence of TNF-α there was no binding ofanti-PEG antibody.

EXAMPLE 9 PEGylation on a Histidine Sequence of Interferon Alpha-2b (IFNα-2b) Possessing an 8 Histidine Sequence on its N-Terminal Using 20 kDaPEG Reagent 9. The Anti-Viral Activity of the 20 kDa PEGylated IFN withand without Reduction with Sodium Borohydride

To a 4.67 ml solution of IFN α-2b with a 8 histidine sequence on theN-terminal (1.07 mg/ml in 50 mM sodium phosphate buffer containing 150mM NaCl, pH 7.4) was added 2.6 molar equivalents of 20 kDa PEG reagent 9(217 μl of a 60 mg/ml solution in deionised water) and the resultingsolution incubated overnight at room temperature. The reaction sampleafter analysis by SDS-PAGE (NuPAGE® Novex 4-12% Bis-Tris gels, MESrunning buffer, all from Invitrogen, and InstantBlue stain (Expedeoncat. No. ISB1L)) was subjected to high performance anion exchangechromatography (TOSOH DEAE column, S TSKgel DEAE-5PW, Supelco Cat No.8-07164, connected to a Jasco HPLC system) to purify the monoPEGylatedIFN α-2b. The fractions obtained (1 ml each) from the ion exchangecolumn were analysed by SDS-PAGE. The fractions containing predominantlymonoPEGylated IFN were subjected to lyophilisation. The resultantresidue obtained after lyophilisation was dissolved in 50 mM sodiumphosphate buffer containing 150 mM NaCl and 2 mM sodium borohydride andleft still at room temperature for 1 h. The resultant solution was thensubjected to size exclusion chromatography (HiLoad 16/60, Superdex 200,50 mM PBS as eluent, 1.6 ml/min flow rate and detection at 214 nm) toisolate the monoPEGylated IFN. The sample isolated was analysed bySDS-PAGE and the result is shown in FIG. 12. In the lane labelled 1 ofFIG. 12 are the protein markers. The lane labelled 2 shows the startingIFN only. Lane 3 shows a single band between the 50 and 60 kDa proteinmarkers corresponding to the purified 20 kDa monoPEGylated IFN α-2b.

Antiviral activity: Antiviral assay was performed on A549 cells culturedin DMEM/10% fetal calf serum (FCS) containing penicillin andstreptomycin. A549 cells were resuspended in DMEM/10% FCS at aconcentration of 0.2×106 cells/ml and aliquoted at 50 μl/well into96-well microtitre plates. The next day, PEGylated and native IFNsamples were prepared in a 2-fold dilution, and 50 μl of each dilutionwas added to the wells. The plates were then incubated for 24 h. Themedia was then removed and the cells were inoculated withencephalomyocarditis virus (EMCV) prepared in DMEM/5% FCS (50 μl/well).Cells were incubated for further 24 h, then washed with 300 μl/well ofPBS and stained with 4% formaldehyde/0.1% methyl violet (50 μl/well;Sigma-Aldrich, cat no. 198099) for 30 min. The plate was then washedtwice with 300 μl/well of PBS and air-dried. The dye was solubilizedwith a 2% SDS solution (50 μl/well) and the absorbance was measured at570 nm. The 20 kDa PEGylated IFN-α2b (A) untreated or (B) treated withsodium borohydride showed activity of 64 pg/ml and 68 pg/ml,respectively.

EXAMPLE 10 PEG Reagent 9 Stability Compared to Commercial PEG Reagentswith Maleimide Functional Groups

The stability of PEG reagent 9 (5 kDa and 20 kDa samples) was comparedto methoxy polyethylene glycol) maleimido-propionamide (ChirotechTechnology Ltd, cat. no. 008-008, lot no. 223126001) andα-methoxy-ω-ethyl-maleinimide polyethylene glycol (Iris Biotech cat. no.PEG1146, lot no. 128512) by nuclear magnetic resonance (NMR)spectroscopy at pH 7.4. A pH 7.4 D₂O solution was made by firstfreeze-drying a 50 mM sodium phosphate aqueous solution containing 150mM NaCl and 20 mM EDTA at pH 7.4 and reconstituting to the same volumewith deuterium oxide. Acetone was added as a standard to the buffer at1.0 μl per 3 ml. Samples of the PEG reagents were dissolved at 1 μmol in0.75 ml of the buffer and analysed after 4 h and after 25.5 h by 400 MHzNMR spectroscopy. The stability of the PEG maleimide samples wasdetermined by comparing the integral at 6.86 ppm after normalising usingthe integral for the acetone standard at 2.17 ppm. The stability of PEGreagent 9 was determined by comparing the total integral for the signalsat 7.31, 7.40, 7.47, 7.73, and 8.03 ppm after normalising using theintegral for the acetone standard at 2.21 ppm. The peaks at 7.31, 7.47and 8.03 are from the protein active PEG reagent 10 (FIG. 14) formed asexpected from PEG reagent 9. The ratio of 9 to 10 ranged from 1.46 to1.77 to 1 for both sample 1 and 2 during the course of the experiment.The stability study results are shown in Table 1. The PEG maleimidesamples degraded by between 19 and 55% over 21.5 h at pH 7.4 while thePEG reagent 9 samples did not degrade under the same conditions.

TABLE 1 Results for a NMR stability study comparing PEG reagent 9 withPEG maleimide. 25.5 h Integral % Degradation 4 h Integral for for 6.86ppm over 21.5 h at Sample 6.86 ppm peak peak pH 7.4 PEG 1.16 0.52 55maleimide sample 1 PEG 0.75 0.61 19 maleimide sample 2 4 h Total 25.5 hTotal Integral for Integral for 7.31, 7.40, 7.31, 7.40, 7.47, 7.73, 8.03ppm 7.47, 7.73, 8.03 peaks ppm peaks PEG reagent 4.85 4.82 <1 9 sample 1PEG reagent 3.93 4.01 0 9 sample 2

EXAMPLE 11 PEGylation of Laminin Fragment 925-983 (Lamβ1₉₂₅₋₉₃₃)Containing a Single Free Cysteine Thiol at Varying pH

Lamβ1₉₂₅₋₉₃₃ is a synthetic linear nonapeptide(Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH₂) (Sigma cat. no. C0668), whichcorresponds to residues 925-933 of the laminin B1 chain and contains asingle cysteine thiol.

PEGylation with PEG Reagent 9 at Varying pH:

Lyophilised Lamβ1₉₂₅₋₉₃₃ peptide (1 mg) was dissolved into 500 μl ofdeionised water to give a 2 mg/ml stock solution. After vortexing, thepeptide stock solution was used fresh or aliquoted and stored at −80° C.until use. The Lamβ1₉₂₅₋₉₃₃ stock solution was diluted two-fold to givea final concentration of 1 mg/ml (1 mM) in the PEGylation reactionmixture. The 5 kDa PEG reagent 9 (5 mg) was dissolved into 200 μl ofdeionised water to give a 25 mg/ml stock solution. After vortexing, thePEG reagent stock solution was used fresh or aliquoted and stored at−80° C. until use. The PEG stock solution was diluted five-fold to givea final concentration of 5 mg/ml (1 mM) in the PEGylation reactionmixture. The buffer stock solution contained 1 M sodium phosphate bufferfor the pH 6.0-8.0 range and 1 M sodium carbonate-bicarbonate buffer forthe pH 8.5-10.0 range. For all pH values, the buffer stock solution alsocontained 10 mM EDTA and 381 μM hydroquinone. The buffer stock solutionwas diluted ten-fold to give a final concentration of 100 mM bufferingagent (sodium phosphate or sodium carbonate-bicarbonate), 1 mM EDTA and38 μM hydroquinone.

Each PEGylation reaction mixture contained 5 μl Lamβ1₉₂₅₋₉₃₃ stocksolution, 1 μl buffer solution, 2 μl of PEG reagent solution(corresponding to a 1:1 molar ration of PEG reagent to Lamβ1₉₂₅₋₉₃₃model peptide) and 2 μl of deionised water to give a total reactionvolume of 10 μl. After vortexing for several seconds, followed by briefcentrifugation (30 s at 5,000×g) to collect the solution at the bottomof the tube, the reaction mixtures were incubated standing at roomtemperature for 0.25, 0.5, 1, 2, 4, 16 and 24 hours. After incubation atroom temperature, the tubes containing the reaction mixtures were placedat −80° C. and stored until analysis by reverse-phase chromatography.

For the reverse-phase chromatography assay a Source 5RPC ST 4.6/150 (GEHealthcare, cat. no. 17-5116-01) column was used. The column wasconnected to a Jasco HPLC system comprising a Jasco PU-980 IntelligentHPLC Pump, a Jasco LG-980-02 Ternary Gradient Unit, a Jasco DegassysPopulaire Degassing Unit, a Jasco UV-970 4-λ Intelligent UV Detector, AJasco LC-NetII/ADC Interface for connection to a PC and a Rheodyne 7725imanual injector valve. The HPLC system was controlled through a computerusing the EZchrom SI version 3.2.1 Build 3.2.1.34 chromatographysoftware package (Agilent Technologies). Chromatogram analysis and dataexport were also performed using the EZchrom SI chromatography datasystem.

A three-eluent system was used. Eluent A contained 5% acetonitrile (FarUV, HPLC grade, Fisher cat. no. A/0627/17) and 0.065% trifluoroaceticacid (TFA) (Acros cat. no. 139721000) in deionised water. Eluent Bcontained 0.075% TFA in acetonitrile, while Eluent C was 100%acetonitrile. Eluents were degassed by sonication before use. Theelution program involved a 0-64% B gradient in 20 minutes, followed bywash in 100% acetonitrile and re-equilibration in eluent A (Table 2). Aconstant flow rate of 1 ml/min was maintained throughout the run. Theabsorbance at 215, 250, 280 and 350 nm was recorded throughout the run.Each sample (10 μl) was thawed and briefly centrifuged (1 min at 14,000g) immediately before 5 μl of supernatant were injection into thereverse phase chromatography column.

TABLE 2 Settings for the gradient elution program used for the reversephase chromatography assay Time (min) Flow rate (ml · min⁻¹) A (%) B (%)C (%) initial 1.00 100 0 0 20.00 1.00 36 64 0 20.20 1.00 0 0 100 24.001.00 0 0 100 24.20 1.00 100 0 0 30.00 1.00 100 0 0

The identity of each peak in the chromatograms was confirmed by runningstandard samples (PEG reagent, reduced and oxidised unreacted peptide)and by analytical size exclusion chromatography (SEC) for relative sizeestimation (for PEGylation product: peptide-PEG conjugate). The columnused for analytical SEC was a BioSep-SEC-S3000 (300×7.8 mm) analyticalcolumn (Phenomenex, cat. no. 00H-2146-KO). The running eluent used was10 mM sodium phosphate buffer (pH 7.0) containing 10% (v/v) acetonitrileand the flow rate was kept constant at 2 ml/min throughout the run.

FIG. 13 shows chromatograms of a time-course PEGylation experiment at pH6.5, using 1 molar equivalent of 5 kDa PEG reagent 9. After 15 minutesincubation at room temperature, five peaks are visible: the PEG reactivepeptide Lamβ1₉₂₅₋₉₃₃ (peak 1); an unreactive peptide dimer formedthrough disulfide formation (peak 2); the PEG reagent 9 sulfonic acidleaving group (peak 3, compound 11 in FIG. 14); a peak corresponding toPEGylated peptide product (5 kDa PEG-Lamβ1₉₂₅₋₉₃₃ conjugate) (peak 4)and the PEG reagent 9 (peak 6, unreactive form).

After 4 hours incubation, all the free peptide (peak 1) had beenconsumed as the PEGylation went to completion. Some excess activated PEGreagent peak (peak 5, compound 10 in FIG. 14) is also present becausesome of the peptide formed an unreactive dimer (peak 2, note that thePEG reagent absorbs more strongly than the peptide at equivalentconcentrations). The oxidised Lamβ1₉₂₅₋₉₃₃ (peak 2) could not bePEGylated as it possesses no free thiol and hence its corresponding peak(peak 2) remained unchanged during the course of the experiment. Thisresult is consistent with cysteine thiol PEGylation occurring for thereduced peptide.

The reactivity of PEG reagent 1 towards Lamβ1₉₂₅₋₉₃₃ between pH 6.0 andpH 8.0 was assessed by measuring the conversion of peptide (peak 1) to 5kDa PEGylated Lamβ1₉₂₅₋₉₃₃ product (peak 4). The peptide peak resultswere normalised by assigning the value of 100 to the calibrated peakarea corresponding to the total amount of peptide used in eachPEGylation reaction, while the product peak results were normalisedusing a deduced maximal product peak area corresponding to totalconversion of peptide into product. Conversion (%) was defined as theproportion of PEGylated peptide relative to the initial amount ofpeptide used in the reaction. The result is shown in FIG. 15. At pH 6.0,6.5, 7.0, 7.5 and 8.0 all the peptide was PEGylated. The rate to go tocompletion depended on the pH, the higher the pH the faster the rate,and reflects the different rate of formation of the reactive structure10 (FIG. 14) at different pH.

EXAMPLE 12 Comparison of the Reactivity of PEG Reagent 9 and aCommercially Available PEG Maleimide at Varying pH

The reactivity of PEG reagent 9 towards the Lamβ1₉₂₅₋₉₃₃ peptide ofExample 11, was compared to a commercially available thiol reactive PEGreagent, PEG maleimide (O-(2-maleimidoethyl)-Ω′-methyl-polyethyleneglycol 5,000, Fluka cat. no. 63187). To ensure that PEG reagent 9 wasimmediately available for reaction in the experiment time-scale, thereagent was first allowed to form the thiol reactive form (Reagent 10,FIG. 14) by incubating at pH 7.5 (2 hours, 4° C.) and the reagent 10isolated by RP chromatography prior to peptide conjugation. All peptidereactions were carried out with freshly dissolved PEG reagent. Briefly,the PEGylation reaction′ solution (reaction scale 10 μl) contained 10 μgLamβ1₉₂₅₋₉₃₃, 50 μg of PEG (5 kDa) reagent (corresponding to a 1:1 molarratio of PEG reagent to Lamβ1₉₂₅₋₉₃₃ model peptide) in 100 mM sodiumphosphate buffer (pH 6.0-8.0) or 100 mM sodium carbonate-bicarbonate (pH8.5-10.0) and 1 mM EDTA. After incubation at room temperature, thereaction mixtures were analysed by reverse-phase chromatography asdescribed previously in Example 11. The result is shown in FIG. 16. ForPEG reagents 9 (pH 9.0 to 10) and 10 (pH 6.0 to 8.0), full conversion ofthe peptide to the PEGylated form could be achieved between pH 6.0 andpH 10 within 15 minutes.

For the PEG maleimide reagent conversion only reached a maximum of 78.9%after the 15 minutes. At higher pH, the conversion was even lower (51.2at pH 10). The PEG maleimide reaction at pH 8.5 was sampled again after16 h and all the peptide had been consumed.

At all pH values tested therefore, PEG reagents 9 and 10 were moreefficient than the PEG maleimide reagent.

EXAMPLE 13 Stability of a PEG Reagent 9 Peptide Conjugate and Comparisonwith a PEG Maleimide Derived Peptide Conjugate

The stability of purified Lamβ1₉₂₅₋₉₃₃ peptide conjugates made from PEGreagent 9 and PEG maleimide from Example 12 were compared over 12 days.The stability of the conjugates was determined by monitoring the area ofthe PEG-peptide peak using reverse phase chromatography as described inExample 11.

Synthesis of PEGylated Lamβ1₉₂₅₋₉₃₃ with PEG Reagent 9:

The conjugate was prepared using a modified method of Example 11. (pH6.0 using a final peptide concentration of 1.6 mg/ml and 0.4 mgpeptide). The product was isolated by RP-chromatography. The elutionpeak corresponding to the Lamβ1₉₂₅₋₉₃₃-PEG conjugate was collected,lyophilised and subsequently re-suspended in deionised water. Finally, 2mM of sodium borohydride (Acros Organics, cat. no. 200050250) were addedand after the sample was incubated for 30 minutes at room temperature,50 mM of sodium phosphate buffer (pH 7.5) was added. Synthesis ofPEGylated Lamβ1₉₂₅₋₉₃₃ with PEG maleimide: Lamβ1₉₂₅₋₉₃₃ (0.4 mg) wasused for PEGylation with 5 kDa PEG maleimide (5 kDa, Fluka cat no.63187) reagent. Briefly, 20 μl of 10× buffer stock solution (containing1 M sodium phosphate buffer (pH 8.0) and 10 mM EDTA) and 40 μl of PEGreagent stock solution (25 mg/ml in deionised water) were added to 200μl of peptide stock solution (2 mg/ml in deionised water). After 1.5hours incubation at room temperature, the product was purified using asRP chromatography as described in Example 11. The elution peak samplewas lyophilised and subsequently re-suspended in deionised water andthen 50 mM of sodium phosphate buffer was added to adjust the pH to 8.0.RP chromatography was used to make sure the concentrations of bothPEG-peptide solutions were approximately equal.

Both stability samples were incubated at room temperature. After 12days, samples were analysed by analytical RPC and the stabilitydetermined by the change in the area of integration for the conjugatepeaks as shown in FIG. 17. The conjugate derived from PEG reagent 9remained stable over 12 days. For the conjugate derived from PEGmaleimide, only 63% remained in the chromatogram showing that PEGreagent 9 lead to a more stable product.

EXAMPLE 14 PEGylation Using PEG Functionalised at Both Ends—PEG Reagent12

PEG reagent 12 was prepared in an analogous way to PEG reagent 9 usingO,O′-bis(2-aminoethyl)polyethyleneglycol 6000 (Fluka cat. no. 14504) andallowed to react with the model peptide from Example 11 (Lamβ1₉₂₅₋₉₃₃).Water (6 μl), 2 μl of 10× buffer stock solution (containing 1 M sodiumphosphate buffer (pH 8.0), 381 μM hydroquinone and 10 mM EDTA) and 2 μlPEG reagent 12 (25 mg/ml) reagent were added to 10 μl Lamβ1₉₂₅₋₉₃₃ stocksolution (2 mg/ml in deionised water). This corresponded to a molarratio of 1:0.375 model peptide to PEG reagent. The reaction solution (5μl) was analysed by analytical RPC (as described in Example 11) after2.5 h incubation at room temperature. All components of the reactionsolution eluted as separate peaks: Lamβ1₉₂₅₋₉₃₃ monomer eluted at 8.1min, oxidised Lamβ1₉₂₅₋₉₃₃ dimer eluted at 8.9 min, un-activated PEGreagent eluted at 17.3 min, activated PEG reagent eluted at 15.7 min andthe leaving group 11 at 10.1 min. The product peak eluted at 14.2minutes. After 2.5 h, 65% of the free Lamβ1₉₂₅₋₉₃₃ present (86% reactionon reagent 12) in the reaction solution had been conjugated showing thatboth reactive ends of the PEG reagent 12 undergo reaction and that thisbis-functional reagent can be successfully used to attach one peptidemolecule at both ends of a PEG molecule.

EXAMPLE 15 Use of Poly(1-Vinyl-2-Pyrrolidone) (PVP) as the PolymerComponent: Conjugation of PVP with Lamβ1925-933 Model PeptidePreparation of PVP Reagent (3 Steps):

Step 1: PVP with a Terminal Amine Group:

A pressure tube was charged with cysteamine (0.028 g), dioxane (8 ml)and a magnetic stir bar. After gentle heating to allow a solution toform, the solution was purged with argon at room temperature for 5 min.While still purging, 1-vinyl-2-pyrrolidone (2.0 g) was then added andafter a further 5 min this was followed by2,2′-azobis(2-methylpropionitrile) (0.089 g). After a further 2 min thepressure tube was sealed with a screw cap under argon and placed in anoil bath at 60° C. for 17 h with stirring. After allowing the tube andcontents to cool to room temperature, diethyl ether (15 ml) was addedcausing precipitation of the polymeric product. The liquid phase wasdecanted away and the solid residue redissolved in acetone (3 ml). Theresulting acetone solution was then added dropwise to rapidly stirringdiethyl ether (25 ml) and the precipitate isolated on a no. 2 sinteredglass funnel with a slight burst of vacuum. The solid was washed withfresh diethyl ether (10 ml) and then allowed to dry under vacuum at roomtemperature (mass=1.44 g, white solid).

Step 2—Conjugation of Protein Reactive End Group to PVP-Amine:

PVP-amine (500 mg), 4-(3-tosylpropanoyl)benzoic acid (structure 6 inFIG. 3, 166 mg), and 4-dimethylaminopyridine (6 mg) were mixed withanhydrous dichloromethane (10 ml) under argon and with stirring was thenadded 1,3-diisopropylcarbodiimide (155 μl). The resulting mixture wasallowed to stir over a weekend at room temperature. Volatiles evaporatedduring the weekend so the solid residue was redissolved indichloromethane (10 ml) and then filtered though non-absorbentcotton-wool. To the filtrate was then added diethyl ether (30 ml) andthe resulting precipitate isolated by centrifugation (4,600 rpm, −9° C.,10 min). The liquid phase was decanted off and the remaining residueredissolved in dichloromethane (10 ml). The diethyl ether precipitationpurification method was repeated twice more and the residue allowed todry under vacuum (513 mg). Diagnostic NMR signals for the PVP conjugatedlinker group occurred at 7.97, 7.82, 7.59 and 7.38 in CDCl₃.

Step 3—Fractionation of PVP Reagent:

A portion (120 mg) of the solid material obtained from the above stepwas mixed with aqueous 20 mM sodium acetate buffer, 150 mM NaCl, pH 4.0and then centrifuged at 13,000 rpm until a clear solution was visibleand the liquid phase 0.45 μm filtered. The filtrate was thenfractionated (1.9 ml loaded) on a HiLoad 16/60 Superdex™ 200 prep gradesize exclusion column (GE Healthcare) running 20 mM sodium acetatebuffer, 150 mM, pH 4.0 at 1 ml/min by collecting fractions every 1 minduring peak elution. The fraction eluting between 73.9 to 74.9 min wasused for protein conjugation after freeze-drying. The peptide reactionwas carried out in 100 mM sodium phosphate buffer (pH 8.0) containing 1mM EDTA, 38 μM hydroquinone, 1.03 mM Lamβ1₉₂₅₋₉₃₃ and an excess of PVPreagent. The reaction mixture was analysed by analytical. RPC asdescribed in Example 11. After one hour incubation at room temperature,approximately 60% of the free Lamβ1₉₂₅₋₉₃₃ model peptide present in thereaction mixture was converted into Lamβ1₉₂₅₋₉₃₃-PVP conjugate (RPCretention time of 11.3 min) successfully demonstrating that polymersother than PEG can be used.

EXAMPLE 16 Synthesis and Use of a Peg Reagent with an Amine-BasedLeaving Group L

Synthesis of PEG Reagent 13:

PEG reagent 13 (FIG. 19) was prepared from the direct conjugation ofcompound 2 (26 mg) with 5 kDa mPEG-NH2 (40 mg) in DMSO (5 ml) using DIPC(11 mg) in an analogous procedure to that of Example 1 to afford anoff-white solid (31 mg). The NMR spectrum of the product (in CDCl₃) gavediagnostic signals at 8.02 and 7.59 ppm.

Reaction with Lamβ1₉₂₅₋₉₃₃ Model Peptide of Example 11:

The reaction was carried out in 100 mM sodium phosphate buffer (pH 7.5)containing 1 mM EDTA, 38 μM hydroquinone, 1.03 mM Lamβ1₉₂₅₋₉₃₃ and 3molar equivalents of PEG reagent 13. The reaction mixture was analysedby analytical RPC as described in Example 11. Monomeric Lamβ1₉₂₅₋₉₃₃eluted at 8.1 min, dimerised Lamβ1₉₂₅₋₉₃₃ eluted at 8.9 min, the amineleaving group eluted at 9.3 min, PEG reagent 13 without the amineleaving group at 15.2 min, PEG reagent 13 at 15.75 min and the productpeak at 14.2 min. After 1 hour incubation at room temperature,approximately 10% of the free Lamβ1₉₂₅₋₉₃₃ model peptide present in thereaction mixture was converted into Lamβ1₉₂₅₋₉₃₃-PEG conjugate.

EXAMPLE 17 The Thiol PEGylation and Activity of IL-1-Ra Using PEGReagent 9

A recombinant nonglycosylated form of the human interleukin-1 receptorantagonist (IL-1-Ra) was used as a model protein. The IL-1-Ra consistsof 153 amino acids, two disulfide bonds (Cys69/Cys116 and Cys66/Cys122),has a molecular weight, of 17.3 kDa and contains an N-terminalhexahistidine tag.

A series of thiol PEGylations were carried out using 1-4 molarequivalents of 10 kDa PEG reagent 9 in 50 mM sodium phosphate buffer (pH6.0 and 7.5). Because the protein was expressed reduced, reduction ofthe disulfides prior to PEGylation was not necessary. The proteinconcentration was 0.1 mg/ml. After 1 hour incubation at 4° C., sampleswere taken for SDS-PAGE analysis and the result is shown in FIG. 18. Atboth pH 6.0 and 7.5, the main product of the PEGylation reaction wasmono-PEGylated IL-1-Ra when 1 molar equivalent of PEG reagent was used.A faint band corresponding to a di-PEGylated product can also be seen,as well as a band corresponding to un-PEGylated IL1-Ra. Increasing themolar equivalents of PEG used in the reaction resulted in an increase indi-, tri- and tetra-PEGylated species. A larger scale PEGylation at pH6.0 was then performed using 1.5 mg IR-1-Ra (0.2 mg/ml) and 1.5 molarequivalents of 20 kDa PEG reagent 9 in 50 mM sodium phosphate buffer pH6.0 containing 20 mM EDTA. The solution was briefly vortexed and placedat 4° C. for 2 hours. The PEGylated IL-1-Ra was purified by immobilisedmetal affinity chromatography (IMAC) and size exclusion chromatography.Prior to IMAC, EDTA was removed by repeated cycles of concentration anddilution with fresh phosphate buffer pH 7.4 using an Amicon ultra-43,000 Da MWCO ultrafiltration centrifugal device (Millipore, cat. no.UFC 800324). Finally, the sample was concentrated to 4 ml and treatedwith 2 mM sodium borohydride for 30 minutes at room temperature followedby loading onto a HisTrap HP (1 ml) column (GE Healthcare cat. no.17-5247-01) pre-equilibrated with phosphate buffer saline (PBS) pH 7.4.The column was washed with 20 ml PBS and elution involved a gradientfrom PBS to PBS containing 500 mM imidazole in 40 minutes. Fractions (1ml) of eluate were collected and an aliquot of each fraction wasanalysed by SDS-PAGE to identify the fractions containing the PEGylatedproduct. Subsequently, the fractions containing the PEGylated productwere pooled together and concentrated by ultrafiltration (Amicon ultra-43,000 Da MWCO) to a final volume of 0.6 ml. The concentrated IMACfractions were loaded onto a HiLoad Superdex 200 16/600 column (GEHealthcare, cat. no. 17-1069-01) which was pre-equilibrated with PBS pH7.4. The flow rate was maintained at 1 ml/min through the run and themono-PEGylated products eluted at about 62 min. Fractions (1 ml) werecollected around the elution peak and an aliquot of each fraction wasanalysed by SDS-PAGE. The fractions containing pure mono-PEGylatedproduct were concentrated by ultrafiltration and re-analysed by SDS-PAGEto confirm purity and the result is shown in Lane 2 of FIG. 19. The gelshows a single band corresponding to mono-PEGylated protein and no freeprotein is visible.

After quantification by UV spectrophotometry, the sample was analysedfor in vitro biological activity by assessing the inhibition ofIL-1β-dependent IL-6 release in MG-63 cells. MG-63 cells were added at20,000 cells/well in 100 μl of DMEM/10% FCS. On the following day,medium was removed and 50 μl/well of fresh medium was added. Sampleswere added in a 5-fold dilution in duplicates (25 μl/well),pre-incubated for 1 h then IL-1β added at a final concentration of 0.3ng/ml (25 μl/ml). The plate was incubated for a further 24 h. Forassessing cell viability, 10 μl of thiozolyl blue tetrazolium bromide(MTT; 5 mg/ml in DMEM; Sigma-Aldrich cat no. M5655) was added to eachwell and incubated for 3 h. The plate was then centrifuged at 1,500 gfor 5 min and the medium was carefully aspirated. The formazan productin metabolically active cells was then dissolved in DMSO (100 μl/well)and the absorbance was measured at 570 nm. The result is shown in FIG.20 and shows that PEGylated IL-1Ra retained inhibitory activity afterPEGylation with reagent 9.

EXAMPLE 18 PEGylation on a Polyhistidine Sequence of IL-1Ra Using PEGReagent 9, and Activity of the PEGylated Protein

The IL-1Ra of Example 17 was also PEGylated on the polyhistidine tagafter oxidising the protein free cysteines to disulfides with coppersulfate prior to the PEGylation reaction as follows: Copper sulfate (1mM) was added to 2.5 ml solution of IL-1Ra (0.6 mg/ml, 1.5 mg IL-1-Ra)in 50 mM Tris.HCl (pH 8.0) containing 200 mM NaCl. After incubation for16 hours at 4° C., 25 mM EDTA was added and sample was loaded on to aPD-10 column (GE Healthcare) pre-equilibrated with 50 mM sodiumphosphate buffer pH 7.5 containing 20 mM EDTA. The column was theneluted with 3.5 ml of fresh phosphate buffer. The protein concentrationused for the PEGylation was 0.43 mg/ml and the pH 7.4 and incubated for16 hours at 4° C. PEG reagent 9 (20 kDa) at 1.5 molar equivalents toprotein. The reaction was incubated for 16 h at 4° C. The monoPEGylatedspecies was isolated using the same chromatography as described inExample 17 along with the sodium borohydride treatment. The SDS-PAGEresult of the product is shown in lane 2 of FIG. 19 where no freeprotein can be seen. The bioactivity of the product was assessed bymeasuring the inhibition of IL-1β-dependent IL-6 release in MG-63 cellsas described in Example 17 and the result is shown in FIG. 20. ThePEGylated IL-1Ra possessed inhibitory activity in the assay.

EXAMPLE 19 PEG Reagent Synthesis: Synthesis of 10 kDa PEG Reagent 15

PEGylated 4-(3-(2-hydroxyethylsulfonyl)propanoyl)benzoic acid reagent 15was prepared from 4-(3-(2-hydroxyethylsulfonyl)propanoyl)benzoic acid 14in an analogous way to that described for the synthesis of PEGylated4-(3-tosylpropanoyl)benzoic acid 9 in Example 1, as shown in FIG. 21.Firstly, 4-(3-(2-hydroxyethylsulfonyl)propanoyl)benzoic acid 14 wasprepared in an analogous way to 4-(3-tosylpropanoyl)benzoic acid butusing mercaptoethanol instead of 4-methylbenzenethiol (Step 2, Example1). NMR 14 (400 MHz) δ 3.35 (t, 2H, COCH₂), 3.5 (overlapping m, 4H,CH₂SO₂CH₂), 3.8 (t, 2H, CH₂OH), 5.2 (br s, CH₂OH), 8.05 (m, 4H, aromaticCH), 13.4 (br s, 1H, COOH). For the PEG conjugation step, the sulfone 14(143 mg) and 0-(2-aminoethyl)-O′-methyl-PEG (MW 10 kDa, 1 g, BioVectra)were dissolved in dry toluene (5 ml). The solvent was removed undervacuum without heating and the dry solid residue was then redissolved indry dichloromethane (10 ml) under argon. To the resulting solution,cooled in an ice bath, was slowly added diisopropylcarbodiimide (DIPC,87 mg) under argon. The reaction mixture was then kept stirring at roomtemperature overnight (15 h). Volatiles were then removed under vacuum(30° C., water bath) to afford a solid residue that was redissolved withgentle heating (35° C.) in acetone (45 ml). The solution was filteredover non-absorbent cotton wool to remove insoluble material. Thesolution was then cooled in a dry ice bath to give a white precipitatethat was separated by centrifugation (4600 rpm, 30 min). The liquidphase was decanted off and this precipitation procedure was repeatedthree times. Afterwards the resulting off-white solid was dried undervacuum to afford the PEG reagent 15 (1 g). ¹H NMR (400 MHz, CDCl₃) δ3.30 (t, 2H, COCH₂), δ 3.40 (s, 3H, PEG-OCH₃), δ 3.40-3.85 (br m, PEG),δ 3.50 (overlapping m, 2×2H, CH₂SO₂CH₂), δ 3.80 (t, 2H, CH₂OH), δ 7.95,δ 8.05 (2×d, 2×2H, ArH of carboxylic acid moiety).

Analogous PEG reagents of different PEG molecular weights were preparedby the same general procedure. Thus, 5 kDa PEG was prepared by reactionof the sulfone 14 (256 mg), O-(2-aminoethyl)-O′-methyl-PEG (5 kDa, 1 g,Biovectra) and DIPC (174 mg) in dry dichloromethane (15 ml) affordingafter the acetone precipitation purification procedure an off-whitesolid 15 (1 g).

EXAMPLE 20 PEG Reagent Synthesis: Synthesis of 10 kDa PEG Reagent 17

PEG reagent 17 was prepared from PEG reagent 9 as follows (FIG. 22): PEGreagent 9 (10 kDa, 75 mg) was allowed to stir with mercaptosuccinic acid(6 mg) and sodium hydrogen carbonate (20 mg) in deionised water (2 ml)for approximately 18 h. Volatiles were then removed on a rotaryevaporator and the solid residue redissolved in warm acetone (4 ml) withan insolubles removed by filtration through non-absorbent cotton-wool.The product was then precipitated from the acetone by cooling thesolution in a dry-ice bath and then isolated by decanting off the liquidphase following centrifugation. The acetone precipitation was repeated afurther three times and the solid was then dried under vacuum to give 16(51 mg). Compound 16 (40 mg) was then oxidised to the sulfone form 17 bymixing with oxone in 1:1 methanol:water (1 ml) for approximately 18 h.The mixture was then diluted with acetone (10 ml) and insolubles werethen removed by filtration under gravity through non-absorbentcotton-wool. The homogeneous filtrate was evaporated to dryness on arotary evaporator and then redissolved in acetone (2 ml) with 2 drops of1 N HCl added and then isolated by a single acetone precipitation asdescribed for 16 (mg). ¹H NMR (400 MHz, CDCl₃) δ 3.24-3.08 (overlappingm, CH₂CH, COCH₂), 3.38 (s, PEG-OCH₃), 3.44 to 3.84 (br s, overlapping m,PEG & CH₂SO₂), 4.44 (dd, SO₂CH), 7.45 (br s, NH), 7.98 & 8.06 (2×d, 4H,ArH of carboxylic acid moiety).

1. A compound of the general formula:X—[NR—CO—Ar—CO—(CH═CH)_(n)—(CH₂)₂-L]_(m)  (I) in which X represents apolymer; R represents a hydrogen atom or an alkyl, aryl or alkyl-arylgroup; Ar represents an unsubstituted or substituted heteroaryl or arylgroup; n represents 0 or an integer of from 1 to 4; L represents aleaving group; and m represents an integer of from 1 to
 8. 2. A compoundas claimed in claim 1, in which X is a polyalkylene glycol,polyvinylpyrrolidone, polyacrylate, polymethacrylate, polyoxazoline,polyvinylalcohol, polyacrylamide, polymethacrylamide, HPMA copolymer,polyester, polyacetal, poly(ortho ester), polycarbonate, poly(iminocarbonate), polyamide, copolymer of divinylether-maleic anhydride andstyrene-maleic anhydride, polysaccharide, or protein.
 3. A compound asclaimed in claim 2, in which X is a polyalkylene glycol.
 4. A compoundas claimed in claim 3, in which X is a polyethylene glycol.
 5. Acompound as claimed in claim 1, in which n is
 0. 6. A compound asclaimed in claim 1, in which L represents —SR′, —SO₂R′, —OSO₂R′, —N⁺R′₃,—N⁺HR′₂, —N⁺H₂R′, halogen, or —OØ, in which R′ represents a hydrogenatom or an alkyl, aryl or alkyl-aryl group and Ø represents asubstituted aryl group containing at least one electron withdrawingsubstituent.
 7. A compound as claimed in claim 6, in which L represents—SO₂R′ in which R′ represents a p-tolyl group.
 8. A compound as claimedin claim 7, in which X is a polyethylene glycol.
 9. A compound asclaimed in claim 1, in which Ar represents a phenyl groupoptionally-substituted by one or more of the same or differentsubstituents selected from the group consisting of alkyl, —CN, —NO₂,—CO₂R″, —COH, —CH₂OH, —COR″, —OR″, —OCOR″, —OCO₂R″, —SR″, —SOR″, —SO₂R″,—NHCOR″, —NR″COR″, —NHCO₂R″, —NR″.CO₂R″, —NO, —NHOH, —NR″.OH,—C═N—NHCOR″, —C═N—NR″.COR″, —N⁺R″₃, —N⁺H₃, —N⁺HR″₂, —N⁺H₂R″, halogen,—C≡CR″, —C═CR″₂, and —C═CHR″, in which each R″ independently representsa hydrogen atom or an alkyl, aryl, or alkyl-aryl group.
 10. A compoundas claimed in claim 9, in which Ar represents an unsubstituted phenylgroup.
 11. A compound as claimed in claim 10, in which X is apolyethylene glycol.
 12. A compound as claimed in claim 1, in which thecompound has the general formula:X—[NH—CO—Ar—CO—(CH═CH)_(n)—(CH₂)₂—SO₂R′]_(m)  (Ia) in which R′represents a hydrogen atom or an unsubstituted or substituted alkyl,aryl or alkyl-aryl group.
 13. A compound as claimed in claim 12, inwhich Ar represents an unsubstituted phenyl group.
 14. A compound asclaimed in claim 13, in which X is a polyethylene glycol.
 15. A compoundas claimed in claim 12, in which n represents
 0. 16. A compound asclaimed in claim 12, in which m represents
 1. 17. A compound as claimedin claim 12, in which R′ represents a p-tolyl group.
 18. A compound asclaimed in claim 12, having the formula:X—NH—CO—Ar—CO—(CH₂)₂—SO₂R′ in which X is a polyethylene glycol, Arrepresents an unsubstituted phenyl group, and R′ represents a p-tolylgroup.